Organic Anion Transporter Protein Research Articles

9-Hydroxy-8-oxypalmatine, a novel liver-mediated oxymetabolite of palmatine, alleviates hyperuricemia and kidney inflammation in hyperuricemic mice

Palmatine is a main bioactive alkaloid of Cortex Phellodendri, which has been commonly prescribed for the treatment of hyperuricemia (HUA) in China. The metabolites of palmatine were crucial to its prominent biological activity. 9-Hydroxy-8-oxypalmatine (9-OPAL) is a novel liver-mediated secondary oxymetabolite of palmatine. The current study was to assess the efficacy of 9-OPAL, a novel liver-mediated secondary oxymetabolite of palmatine derived from Cortex Phellodendri, in experimental HUA mouse model and further explore its underlying mechanism. An in vitro metabolic experiment with OPAL was carried out using liver samples. We separated and identified a novel liver metabolite, and investigated its anti-HUA effect in mice. HUA mice were induced by potassium oxonate and hypoxanthine daily for one week. After 1 h of modeling, mice were orally administered with different doses of 9-OPAL (5, 10 and 20 mg/kg). The pathological changes of the kidneys were evaluated using hematoxylin-eosin staining (H&E). The acute toxicity of 9-OPAL was assessed. The effects of 9-OPAL on serum levels of uric acid (UA), adenosine deaminase (ADA), xanthine oxidase (XOD), creatinine (CRE), blood urea nitrogen (BUN) and inflammatory cytokines were measured by enzyme-linked immunosorbent assay (ELISA) or biochemical method. Furthermore, Western blot, quantitative real-time PCR (qRT-PCR) and molecular docking were used to investigate the effect of 9-OPAL on the expression of renal urate transporters and NLRP3 signaling pathway in HUA mice. 9-OPAL had been discovered to be a novel liver-mediated oxymetabolite of palmatine for the first time. Treatment with 9-OPAL significantly reduced the UA, CRE as well as BUN levels, and also effectively attenuated abnormal renal histopathological deterioration with favorable safety profile. Besides, 9-OPAL significantly decreased the serum and hepatic activities of XOD and ADA, dramatically inhibited the up-regulation of UA transporter protein 1 (URAT1) and glucose transporter protein 9 (GLUT9), and reversed the down-regulation of organic anion transporter protein 1 (OAT1). Additionally, 9-OPAL effectively mitigated the renal inflammatory markers (TNF-α, IL-1β, IL-6 and IL-18), and downregulated the transcriptional and translational expressions of renal Nod-like receptor family pyrin domain containing 3 (NLRP3), caspase-1, apoptosis-associated speck-like (ASC) and IL-1β in HUA mice. Molecular docking results revealed 9-OPAL bound firmly with XOD, OAT1, GLUT9, URAT1, NLRP3, caspase-1, ASC and IL-1β. 9-OPAL was found to be a novel liver-mediated secondary metabolite of PAL with favorable safety profile. 9-OPAL had eminent anti-hyperuricemic and renal-protective effects, and the mechanisms might be intimately associated with repressing XOD activities, modulating renal urate transporter expression and suppressing the NLRP3 inflammasome activation. Our investigation might also provide further experimental evidence for the traditional application of Cortex Phellodendri in the treatment of HUA.

The impact of SLCO1B1 single nucleotide polymorphism and non-genetic factors on statin-induced myopathy

Abstract Funding Acknowledgements None. Background Statins, also known as HMG CoA Reductase Inhibitors, are a class of drugs considered the drug of choice for treating hypercholesterolemia. Statins are well tolerated; however, they may cause severe myopathy in 20% to 30% of patients, due to the individual variation regarding its efficacy and related adverse effects. Of these, comes the Single Nucleotide Polymorphism (SNP) T521C of the SLCO1B1 gene that encodes the protein involved in the trafficking of statins from the blood to hepatocytes which is called Organic Anion Transport Protein 1B1 (OATP1B1). The T521C single nucleotide polymorphism (SNP) results in a substitute of valine with alanine resulting in a decreased or lack of functional protein. Purpose The aim is to determine the frequency of the high-risk C allele and to define its impact on statin-induced myopathy in Syrian patients on statins. In addition to the non-genetic factors that may affect the ability to develop statin-induced myopathy. Methods This was an observational case-control study that had ethical approval from Damascus University (No.433/ 2020). The case group included 44 patients on statins who experienced myopathy, 18 patients who had increased creatinine kinase levels (CK), and 26 patients with normal CK levels, while the control group encompassed 56 patients who were taking statins and had no muscular adverse events. The DNA was extracted from each sample and then genotyped using standard sequencing of specific PCR products using a specific pair of primers. Results The age of patients in the case arm ranged between 27 and 84 years with an average ± SD of (58.2 ± 12.7 years), 70% of them were males. In comparison with (53.34 ± 12.4 years) in the control arm, 66% of them were males without a statistically significant differences (p=0.672, 0.057) respectively. The statin type and dose have a statistically non-significant effect, the average statin dose in the case arm and the control arm was 26.02 mg and 31.27 mg respectively (p=0.072). While the statin type treatment varied as 59% used atorvastatin and 39% used rosuvastatin in the case group compared with 71% used atorvastatin and 27% used rosuvastatin in the control group, without a statistically significant difference (p=0.252). The frequency of the C allele reaches 19.3% and is distributed in three genotypes: wild-type TT (68%), heterozygote TC (25%), and homozygote CC (7%) in the case arm, while it does not exceed 8% in the control arm with the absence of the CC genotype. Only a statistically significant difference was found in the frequency of the CC genotype between these two groups (p=0.047). Conclusion Our study is the first to identify the frequency of the 521T>C SNP of the SLCO1B1 gene in Syrian patients taking statins. Our data reveals a high prevalence of risk allele C in the patients who suffer from statin-induced myopathy with a statically significant difference from the control arm (p=0.026).

296 Towards Nose-to-brain Inhibition of NFB: Activity of a Novel p65 Inhibitor in Glioblastoma and Feasibility of Intranasal Delivery With a Near-infrared Fluorescent HMC-Chitosan Nanoparticle

INTRODUCTION: Constitutive NFκB inflammatory signaling is a driving force of tumor progression, immune evasion, and therapeutic resistance in glioblastoma (GBM). CRL1101 is a novel small molecule inhibitor of RelA (p65), a transcription factor of NFκB-mediated gene expression. Previously, we have shown that heptamethine carbocyanine (HMC) conjugated nanoparticles target GBM tumor sites via organic anion transporter proteins. The activity of CRL1101 in GBM has not been explored. Poor blood-brain barrier (BBB) penetration and theoretical toxicities related to p65 inhibition pose challenges to its implementation in vivo. METHODS: GBM transcriptomic analysis was performed using Gene Profiling Interactive Analysis. In vitro cytotoxicity and migration assays were performed in U87 and GL261. Western blot was used to confirm the mechanism of CRL1101. Quantitative PCR was used to assess the relative expression of NFκB-regulated genes. HMC-chitosan-CRL1101 nanoparticles were synthesized by cross-linking chitosan with HMC and their uptake was visualized using immunocytochemistry. For survival analysis, we implanted GL261 cells into immunocompetent mice and treated intranasally with HMC-chitosan-CRL1101. RESULTS: RelA is overexpressed in GBM relative to normal brain tissue. CRL1101 alone effectively kills U87 cells (IC50 = 16.0 uM) and prevents migration in vitro (p < 0.0001) by disabling the nuclear translocation of RelA. Treatment with CRL1101 was associated with a decrease in IL-8 (p = 0.01) and PD-L1 (p = 0.44) expression. HMC-chitosan-CRL1101 demonstrated an IC50 of 25.23 uM in U87. Near-infrared imaging confirmed intranasal delivery of HMC-chitosan-CRL1101 across the BBB in vivo. CONCLUSIONS: RelA-targeted NFκB inhibition with CRL1101 is a promising immunotherapeutic strategy in GBM. Intranasal nanoparticle delivery is a practical route to bypass the BBB and minimize systemic toxicities in mouse models of GBM, with expected survival benefit in vivo.

Increased Perfluorooctanesulfonate (PFOS) Toxicity and Accumulation Is Associated with Perturbed Prostaglandin Metabolism and Increased Organic Anion Transport Protein (OATP) Expression.

Perfluorooctanesulfonate (PFOS) is a widespread environmental pollutant with a long half-life and clearly negative outcomes on metabolic diseases such as fatty liver disease and diabetes. Male and female Cyp2b-null and humanized CYP2B6-transgenic (hCYP2B6-Tg) mice were treated with 0, 1, or 10 mg/kg/day PFOS for 21 days, and surprisingly it was found that PFOS was retained at greater concentrations in the serum and liver of hCYP2B6-Tg mice than those of Cyp2b-null mice, with greater differences in the females. Thus, Cyp2b-null and hCYP2B6-Tg mice provide new models for investigating individual mechanisms for PFOS bioaccumulation and toxicity. Overt toxicity was greater in hCYP2B6-Tg mice (especially females) as measured by mortality; however, steatosis occurred more readily in Cyp2b-null mice despite the lower PFOS liver concentrations. Targeted lipidomics and transcriptomics from PFOS-treated Cyp2b-null and hCYP2B6-Tg mouse livers were performed and compared to PFOS retention and serum markers of toxicity using PCA. Several oxylipins, including prostaglandins, thromboxanes, and docosahexaenoic acid metabolites, are associated or inversely associated with PFOS toxicity. Both lipidomics and transcriptomics indicate PFOS toxicity is associated with PPAR activity in all models. GO terms associated with reduced steatosis were sexually dimorphic with lipid metabolism and transport increased in females and circadian rhythm associated genes increased in males. However, we cannot rule out that steatosis was initially protective from PFOS toxicity. Moreover, several transporters are associated with increased retention, probably due to increased uptake. The strongest associations are the organic anion transport proteins (Oatp1a4-6) genes and a long-chain fatty acid transport protein (fatp1), enriched in female hCYP2B6-Tg mice. PFOS uptake was also reduced in cultured murine hepatocytes by OATP inhibitors. The role of OATP1A6 and FATP1 in PFOS transport has not been tested. In summary, Cyp2b-null and hCYP2B6-Tg mice provided unique models for estimating the importance of novel mechanisms in PFOS retention and toxicity.

Influence of dietary bioactive compounds on the bioavailability and excretion of PFOA and its alternative HFPO-TA: Mechanism exploration

Oral ingestion is considered an important route of human exposure to perfluorooctanoic acid (PFOA) and its alternative hexafluoropropylene oxide trimer acid (HFPO-TA). Bioactive compounds are widely used as dietary supplements and food additives. However, little is known about the influence of dietary bioactive compounds on the bioavailability of PFOA and HFPO-TA. Here, three dietary bioactive compounds, β-carotene, quercetin and curcumin, were selected to study their influence on the relative bioavailability (RBA) of PFOA and HFPO-TA in soil using a mouse model. Compared to the control group (68.7 ± 6.27 %), quercetin and curcumin at medium and high doses (20 and 100 mg/kg/d) significantly (p < 0.05) decreased PFOA RBA to 55.2 ± 4.85–56.4 ± 4.57 % and 48.3 ± 5.49–48.6 ± 5.44 %, respectively. Mechanism study showed that medium- and high-dose quercetin as well as high-dose curcumin increased urinary excretion of PFOA by 33.6–35.6 % and 32.2 % through upregulating renal expression of multidrug resistance protein 2 (Mrp2) (1.52–1.63 folds) and Mrp4 (1.26–1.53 folds), thereby reducing PFOA accumulation. In PFOA-treated groups, quercetin at medium and high doses dramatically downregulated the hepatic expression of organic anion transport polypeptides (Oatp1a6, Oatp1b2), organic anion transport proteins (Oat1, Oat2), and fatty acid binding proteins (FABP4, FABP12), while curcumin at medium and high doses inhibited the hepatic expression of Oatp1a6, Oat1 and Oat2. These downregulated genes may reduce the transport of PFOA from blood to liver, and in turn decrease the PFOA RBA. However, β-carotene, quercetin and curcumin exhibited no significant effect on RBA and excretion of HFPO-TA (p > 0.05). This indicated the different absorption mechanisms between PFOA and HFPO-TA, and further research is warranted. This study provided a novel perspective for establishing environmentally friendly ways to reduce health hazards from per- and polyfluoroalkyl substances (PFASs).

Associations among Plasma Concentrations of Edoxaban and M-4, Prothrombin Time, and the SLCO1B1*15 Haplotype in Patients With Nonvalvular Atrial Fibrillation.

The authors aimed to examine the impact of single nucleotide polymorphisms in P-glycoprotein, the hepatic uptake transporter organic anion transporter protein 1B1, cytochrome P450 ( CYP ) 3A5, and carboxylesterase-1 ( CES1 ) on the steady-state dose-adjusted trough concentrations of edoxaban (C Edo /D) and M-4 (C M-4 /D). They also investigated whether C M-4 and C Edo affect prothrombin time (PT). The analyses included 152 patients with nonvalvular atrial fibrillation (NVAF) undergoing AF catheter ablation. The CYP3A5*3 ; CES1 c.1168-33A>C, c.257+885T>C; SLCO1B1 c.388A>G, c.521T>C; and ABCB1 c.3435C>T, c.2677G>A/T, c.1236C>T genotypes were determined. Stepwise selection multiple linear regression analyses demonstrated that creatinine clearance (Ccr), concomitant use of amiodarone, and SLCO1B1*15 haplotype status were independent factors influencing C M-4 /D (partial R2 = 0.189, 0.098, 0.067, respectively, all P values < 0.005). Ccr and concomitant use of amiodarone were independent factors influencing C Edo /D (partial R2 = 0.260, 0.117, respectively, both P value < 0.001). C Edo and C M-4 showed a weak correlation with PT (ρ = 0.369 and 0.315, both P values < 0.001). Although information concerning Ccr, concomitant use of amiodarone, and SLCO1B1*15 haplotype may be useful in assessing the pharmacokinetics of edoxaban, further studies are needed to clarify the requirement of PT monitoring at the trough level for dose adjustment of edoxaban in patients with NVAF.

The role of organic anion transport protein 1a4 in drug delivery and diseases: a review

OATP1A4 is an important member of the family of organic anion transporting polypeptides (OATPs), which is generally thought to mediate cellular uptake of endogenous and exogenous substances, such as bile acids, drugs and environmental toxins. Recent studies have found that Oatp1a4 plays an important role in drug passage through the blood-brain barrier and is expected to be an important target for drugs used to treat central diseases. Oatp1a4 has also been associated with various diseases such as cholestasis. differences in Oatp1a4 across age and sex have also become an area of concern for precision drug administration. Therefore, this paper presents a systematic review of Oatp1a4 expression in drug transport and various physiopathological states.

Effects of rifampin on the pharmacokinetics and pharmacodynamics of milvexian, a potent, selective, oral small molecule factor XIa inhibitor

Milvexian (BMS-986177/JNJ-70033093) is a potent, oral small molecule that inhibits the active form of factor XI with high affinity and selectivity. This study assessed the single-dose pharmacokinetic and pharmacodynamic properties of milvexian co-administered with rifampin, an organic anion transport protein (OATP) inhibitor and potent cytochrome P450 (CYP) 3A and P-glycoprotein (P-gp) inducer. In this open-label, nonrandomized, single-sequence study, healthy participants (N = 16) received single doses of milvexian on Day 1 (100 mg), milvexian and rifampin (600 mg) on Day 4, rifampin on Days 5–11, milvexian and rifampin on Day 12, and rifampin on Days 13–14. Pharmacokinetic data were summarized using descriptive statistics. Administration of milvexian, alone or in combination with rifampin, was generally safe and well tolerated. Single-dose co-administration of rifampin and milvexian demonstrated no meaningful changes in milvexian exposure versus milvexian alone (Cmax, 110%; AUC[0–T], 102%; AUC[INF], 101%). After multiple doses of rifampin and milvexian, peak and total milvexian exposure substantially decreased versus milvexian alone (Cmax, 22%; AUC[0–T], 15%; AUC[INF], 15%). Results were consistent with preclinical data, indicating that milvexian is a substrate for CYP3A4/5 and P-gp but not OATP. The implications of these results on the need for dose adjustment of milvexian will be further elucidated following the completion of phase 2 and 3 trials.Trial registration The study was registered with ClinicalTrials.gov (NCT02959060; submitted 7/11/2016, first posted 8/11/2016).

Making high salt concentrations for optimal chromatography compatible with electrospray ionization mass spectrometry using an ion exchange membrane suppressor: Analysis of biomarkers for transporter protein inhibition as a case study

This study reports on the potential of using ion-exchange suppressor technology in liquid chromatography—electrospray ionization mass spectrometry workflows. The aim was to use high salt concentrations to improve separation performance, while overcoming the resulting significant ion suppression during electrospray ionization. As a case study, we apply suppressor technology to the hydrophilic interaction liquid chromatography separation and detection of taurine and glycochenodeoxycholate sulfate, endogenous biomarkers for organic anion transporter protein inhibition. The desired chromatographic selectivity was achieved applying 100 mM ion-pairing agent, while competing ions negatively affecting MS sensitivity were actively removed post-column from the solvent via a charged partially permeable membrane and replaced with protons, resulting in an up to 10-fold increase in detection sensitivity.

Polymorphism of the organic anion transporter protein 1B1 OATP1B1 rs2306283 gene in children with thyroid diseases living in the Far North

Polymorphism of the organic anion transporter protein 1B1 OATP1B1 rs2306283 gene in children with thyroid diseases living in the Far North

Editor’s Letter

<i>SLCO1B1 </i>(<i>solute carrier organic anion transporter family member 1B1</i>) is an important transmembrane hepatic uptake transporter. Genetic variants in the <i>SLCO1B1</i> gene have been associated with altered protein folding, resulting in protein degradation and decreased transporter activity. Next-generation sequencing (NGS) of pharmacogenes is being applied increasingly to associate variation in drug response with genetic sequence variants. However, it is difficult to link variants of unknown significance with functional phenotypes using “one-at-a-time” functional systems. Deep mutational scanning (DMS) using a “landing pad cell–based system” is a high-throughput technique designed to analyze hundreds of gene open reading frame (ORF) missense variants in a parallel and scalable fashion. We have applied DMS to analyze 137 missense variants in the <i>SLCO1B1</i> ORF obtained from the Exome Aggregation Consortium project. ORFs containing these variants were fused to green fluorescent protein and were integrated into “landing pad” cells. Florescence-activated cell sorting was performed to separate the cells into four groups based on fluorescence readout indicating protein expression at the single cell level. NGS was then performed and <i>SLCO1B1</i> variant frequencies were used to determine protein abundance. We found that six variants not previously characterized functionally displayed less than 25% and another 12 displayed approximately 50% of wild-type protein expression. These results were then functionally validated by transporter studies. Severely damaging variants identified by DMS may have clinical relevance for <i>SLCO1B1-</i>dependent drug transport, but we need to exercise caution since the relatively small number of severely damaging variants identified raise questions with regard to the application of DMS to intrinsic membrane proteins such as organic anion transporter protein 1B1. <h3>Significance Statement</h3> The functional implications of a large numbers of open reading frame (ORF) “variants of unknown significance” (VUS) in transporter genes have not been characterized. This study applied deep mutational scanning to determine the functional effects of VUS that have been observed in the ORF of <i>SLCO1B1</i>(<i>s</i><i>olute carrier organic anion transporter family member 1B1</i>). Several severely damaging variants were identified, studied, and validated. These observations have implications for both the application of deep mutational scanning to intrinsic membrane proteins and for the clinical effect of drugs and endogenous compounds transported by <i>SLCO1B1</i>.

SLCO1B1: Application and Limitations of Deep Mutational Scanning for Genomic Missense Variant Function.

SLCO1B1 (solute carrier organic anion transporter family member 1B1) is an important transmembrane hepatic uptake transporter. Genetic variants in the SLCO1B1 gene have been associated with altered protein folding, resulting in protein degradation and decreased transporter activity. Next-generation sequencing (NGS) of pharmacogenes is being applied increasingly to associate variation in drug response with genetic sequence variants. However, it is difficult to link variants of unknown significance with functional phenotypes using “one-at-a-time” functional systems. Deep mutational scanning (DMS) using a “landing pad cell–based system” is a high-throughput technique designed to analyze hundreds of gene open reading frame (ORF) missense variants in a parallel and scalable fashion. We have applied DMS to analyze 137 missense variants in the SLCO1B1 ORF obtained from the Exome Aggregation Consortium project. ORFs containing these variants were fused to green fluorescent protein and were integrated into “landing pad” cells. Florescence-activated cell sorting was performed to separate the cells into four groups based on fluorescence readout indicating protein expression at the single cell level. NGS was then performed and SLCO1B1 variant frequencies were used to determine protein abundance. We found that six variants not previously characterized functionally displayed less than 25% and another 12 displayed approximately 50% of wild-type protein expression. These results were then functionally validated by transporter studies. Severely damaging variants identified by DMS may have clinical relevance for SLCO1B1-dependent drug transport, but we need to exercise caution since the relatively small number of severely damaging variants identified raise questions with regard to the application of DMS to intrinsic membrane proteins such as organic anion transporter protein 1B1.Significance StatementThe functional implications of a large numbers of open reading frame (ORF) “variants of unknown significance” (VUS) in transporter genes have not been characterized. This study applied deep mutational scanning to determine the functional effects of VUS that have been observed in the ORF of SLCO1B1(solute carrier organic anion transporter family member 1B1). Several severely damaging variants were identified, studied, and validated. These observations have implications for both the application of deep mutational scanning to intrinsic membrane proteins and for the clinical effect of drugs and endogenous compounds transported by SLCO1B1.

An 'Omics Approach to Unraveling the Paradoxical Effect of Diet on Perfluorooctanesulfonic Acid (PFOS) and Perfluorononanoic Acid (PFNA)-Induced Hepatic Steatosis.

Perfluoroalkyl substances (PFAS) are a family of toxicants universally detected in human serum and known to cause dyslipidemia in animals and humans. Hepatic steatosis, which is defined as lipid deposition in the liver, is known to be a consequence of poor diet. Similarly, PFAS are known to induce hepatic steatosis in animals on a low-fat chow. This study explored diet-PFAS interactions in the liver and their potential to modulate hepatic steatosis. Male C57BL/6J mice were fed with either a low-fat diet (10% kcal from fat, LFD) or a moderately high-fat diet (45% kcal from fat, HFD) with or without perfluorooctanesulfonic acid (3 ppm, PFOS) or perfluorononanoic acid (3 ppm, PFNA) in feed for 12 weeks. Livers were excised for histology and quantification of PFAS and lipids. The PFOS and PFNA coadministration with HFD reduced the hepatic accumulation of lipid and PFAS relative to the LFD treatment groups. Furthermore, transcriptomic analysis revealed that PFAS administration in the presence of an HFD significantly reduces expression of known hepatic PFAS uptake transporters, organic anion transporter proteins. Transcriptomics and proteomics further revealed several pathways related to lipid metabolism, synthesis, transport, and storage that were modulated by PFAS exposure and further impacted by the presence of dietary fat. Both dietary fat content and the chemical functional head group exerted significant influence on hepatic PFAS accumulation and the resulting biochemical signature, suggesting that diet and structure should be considered in the design and interpretation of research on PFAS induced hepatic steatosis.

Effect of Organic Anion Transporters on the Development of Nephrotoxicity in the Context of NSAIDs Use

Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely used worldwide as pain relievers, antipyretics, and anti-inflammatory drugs. Failure to comply with the instructions for medical use of this group of drugs increases the risk of serious adverse reactions on the part of different organs and systems. From 5 to 18% of patients taking NSAIDs develop adverse reactions associated with impaired renal function. Organic anion transporter (OAT) proteins, which mediate the drug excretion with urine, have an important role to play in the NSAIDs adverse effect on kidneys. The aim of the study was to analyse and systematize scientific literature on the role of OATs in nephrotoxicity development in the context of NSAIDs use. It was revealed that adverse kidney reactions associated with NSAIDs are determined by several mechanisms, including inhibition of prostaglandin synthesis due to cyclooxeganse-1 and/or cyclooxeganse-2 blockade, and direct toxic effect on renal tubule epithelium followed by tubular necrosis due to NSAIDs interaction with OATs. Moreover, by suppressing OAT1 and OAT3, NSAIDs can not only enhance, but also reduce nephrotoxic effects of other medicines (when used together) and endogenous/exogenous toxins. Considering that NSAIDs are widely used in the treatment of various diseases (including in elderly patients and patients with concomitant renal diseases), it is still relevant to study mechanisms of adverse kidney reactions associated with drug transporters.

Polymethine Dye-Functionalized Nanoparticles for Targeting CML Stem Cells

In chronic myelogenous leukemia (CML), treatment with tyrosine kinase inhibitors (TKI) is unable to eradicate leukemic stem cells (LSC). Polymethine dye-functionalized nanoparticles can be internalized by specific cell types using transmembrane carrier proteins. In this study we investigated the uptake behavior of various polymethine dyes on leukemia cell lines and searched for carrier proteins that guide dye transport using RNA interference. The results show that the uptake of DY-635 is dependent on organic anion transport protein 1B3 (OATP1B3) in CML cells and immature myeloid precursor cells of CML patients. In contrast to nonspecific poly(lactide-co-glycolic acid) (PLGA) nanoparticle constructs, DY-635-functionalization of nanoparticles led to an uptake in CML cells. Investigation of these nanoparticles on bone marrow of CML patients showed a preferred uptake in LSC. The transcription of OATP1B3 is known to be induced under hypoxic conditions via the hypoxia-inducing factor 1 alpha (HIF1α), thus also in the stem cells niche. Since these cells have the potential to repopulate the bone marrow after CML treatment discontinuation, eliminating them by means of drug-loaded DY-635-functionalized PLGA nanoparticles deployed as a selective delivery system to LSC is highly relevant to the ongoing search for curative treatment options for CML patients.

Genetic Factors and Delayed TSB Monitoring and Treatment as Risk Factors Associated with Severe Hyperbilirubinemia in Term Neonates Admitted for Phototherapy.

This study aimed to determine whether maternal-fetal blood group isoimmunization, breastfeeding, birth trauma, age when first total serum bilirubin (TSB) was measured, age of admission, and genetic predispositions to hemolysis [due to genetic variants of glucose-6-phosphate dehydrogenase (G6PD) enzyme], and reduced hepatic uptake and/or conjugation of serum bilirubin [due to genetic variants of solute carrier organic anion transporter protein family member 1B1 (SLCO1B1) and uridine diphosphate glucuronosyltransferase family 1 member A1 (UGT1A1)] were significant risk factors associated with severe neonatal hyperbilirubinemia (SNH, TSB ≥ 342µmol/l) in jaundiced term neonates admitted for phototherapy. The inclusion criteria were normal term neonates (gestation ≥ 37 weeks). Parents/care-givers were interviewed to obtain data on demography, clinical problems, feeding practice and age when first TSB was measured. Polymerase chain reaction-restriction fragment length polymorphism method was used to detect common G6PD, UGT1A1 and SLCO1B1 variants on each neonate's dry blood specimens. Of 1121 jaundiced neonates recruited, 232 had SNH. Logistic regression analysis showed that age (in days) when first TSB was measured [adjusted odds ratio (aOR) = 1.395; 95% confidence interval (CI) 1.094-1.779], age (in days) of admission (aOR = 1.127; 95% CI 1.007-1.260) and genetic mutant UGT1A1 promoter A(TA)7TAA (aOR = 4.900; 95% CI 3.103-7.739), UGT1A1 c.686C>A (aOR = 6.095; 95% CI 1.549-23.985), SLCO1B1 c.388G>A (aOR = 1.807; 95% CI 1.242-2.629) and G6PD variants and/or abnormal G6PD screening test (aOR = 2.077; 95% CI 1.025-4.209) were significantly associated with SNH. Genetic predisposition, and delayed measuring first TSB and commencing phototherapy increased risk of SNH.

Chronic Inhibition of CYP3A is Temporarily Reduced by Each Hemodialysis Session in Patients With End-Stage Renal Disease.

Drug dosing is challenging in patients with end-stage renal disease. Not only is renal drug elimination reduced, but nonrenal clearance pathways are also altered. Increasing evidence suggest that uremia impacts drug metabolizing enzymes and transporters leading to changes in nonrenal clearance. However, the exact mechanisms are not yet fully understood, and the acute effects of dialysis are inadequately investigated. We prospectively phenotyped cytochrome P450 3A (CYP3A; midazolam) and P-glycoprotein (P-gp)/organic anion-transporting proteins (OATP; fexofenadine) in 12 patients on chronic intermittent hemodialysis; a day after ("clean") and a day prior to ("dirty") dialysis. Unbound midazolam clearance decreased with time after dialysis; median (range) reduction of 14% (-3% to 41%) from "clean" to "dirty" day (P=0.001). Fexofenadine clearance was not affected by time after dialysis (P=0.68). In conclusion, changes in uremic milieu between dialysis sessions induce a small, direct inhibitory effect on CYP3A activity, but do not alter P-gp/OATP activity.

In Silico Studies to Demonstrate Potential Drug‐Drug Interaction (DDI) By Disrupting Cellular Homeostasis of Organic Anion Transporting Proteins (OATP)

Membrane transporters OATP1B1 and OATP1B3 mediate hepatic uptake of multiple drugs (e.g., statins, antibiotics, antidiabetics). Altered OATP levels or activities cause DDI. The 2012 FDA recommendation for DDI investigations focused on competitive inhibition of OATP using co‐incubation of victim drug (VD) and perpetrator drug (PD). This method has yielded false‐negatives and underestimated severe/fatal adverse events in patients, indicating other yet unknown causes of DDI. The DDI research community named unexpected schedule‐dependent (long pre‐incubation vs. short co‐incubation) DDI as a priority, whereas FDA recommends, in 2017, additional pre‐incubation of VD and PD.We used in silico studies to evaluate if disrupting cellular transporter homeostasis is sufficent to induce clinically significant DDI (defined by FDA as causing &gt;20–25% deviations in VD uptake, measured as area‐under‐concentration‐time curve or AUC), and if the resulting DDI is schedule‐dependent.The figure shows a cellular homeostasis model including biosynthesis and trafficking of OATP in endocytic organelles (from membrane to early endosome (EE) to recycling endosome (RE) and back to membrane, or from EE to late endosome (LE) followed by degradation). The model assumes (a) rapid OATP recycling, (b) rapid OATP degradation after entering LE, (c) slow, zero‐order OATP biosynthesis, (d) VD enters cells through transporter, (e) PD enters or exit cells via passive diffusion, (f) PD stimulates or inhibits the first‐order rate constants of OATP transfer among endocytic organelles, and (g) negligible OATP exocytosis. We used ordinary differential equations (ODE) to depict (a) OATP homeostasis, (b) intracellular pharmacokinetics of VD and PD, and (c) pharmacodynamics of PD‐mediated perturbations (IC50 and Hill coefficient n). Model parameter values were taken from literature reports on other membrane transporters (e.g. internalization of human organic anion transporter 1), calculated, or arbitrarily set based on current knowledge of cellular and endosomal pathways. ODE were solved in MATLAB (ode45 algorithm) to compute the intracellular VD AUC as functions of (a) distribution of OATP in membrane vs. cytosol at baseline (i.e., without PD), (b) extracellular concentrations of VD and PD, (c) OATP‐mediated uptake of VD, and (d) potency of PD on inhibiting or stimulating selected endocytic transfer processes.The in silico results show disruptions of OATP homeostasis induced clinically significant DDI. The extent of DDI increased with VD+PD co‐incubation time, baseline cytosol‐to‐membrane ratio of OATP, extent of PD‐perturbations of kEE and kRE, and difference between kEE and kRE. These results provide a theoretical proof‐of‐concept that perturbing OATP homeostasis may cause clinically significant DDI in a time‐dependent manner.Support or Funding InformationSupported in part by research grants from NCI and NIBIB (R01CA163015, R01EB015253) and FDA (2091IPAAU, 2091IPAWIENTJES), and by Mosier Chair of Pharmaceutical Sciences.Figure 1

Interindividual Diversity in Expression of Organic Anion Uptake Transporters in Normal and Cirrhotic Human Liver.

The liver plays an essential role in removing endogenous and exogenous compounds from the circulation. This function is mediated by specific transporters, including members of the family of organic anion transport proteins (OATPs) and the Na+‐taurocholate transporting polypeptide (NTCP). In the present study, transporter protein expression was determined in liver samples from patients with cirrhosis or controls without liver disease. Five transporters (OATP1A2, OATP1B1, OATP1B3, OATP2B1, and NTCP) were studied. Transporter content in homogenates of human liver was quantified on western blots probed with transporter‐specific antibodies in which a calibrated green fluorescent protein‐tagged transporter standard was included. Liver samples from 21 patients with cirrhosis (hepatitis C in 17 and alcohol abuse in 4) and 17 controls without liver disease were analyzed. Expression of each of the transporters had a large spread, varying by an order of magnitude in cirrhotic and control livers. OATP1B1 was the most abundant transporter in controls (P < 0.01) but was significantly lower in cirrhotic livers as was NTCP expression (P < 0.01). There was little difference in transporter expression with respect to age or sex. Despite the large variability in transporter expression within a group, analysis in individuals showed that those with high or low expression of one transporter had a similar magnitude in expression of the others. Conclusion: Differences in transporter expression could explain unanticipated heterogeneity of drug transport and metabolism in individuals with and without liver disease.

2500. Fostemsavir Drug–Drug Interaction Profile, an Attachment Inhibitor and Oral Prodrug of Temsavir, for Heavily Treatment Experienced HIV-1-Infected Patients

BackgroundFostemsavir (FTR) is a first-in-class attachment inhibitor being evaluated in heavily treatment-experienced (HTE) HIV-1-infected patients. Active temsavir (TMR) binds to viral envelope glycoprotein 120 and prevents viral attachment and entry into host CD4+ T cells. TMR is primarily metabolized by esterase-mediated hydrolysis with contributions from cytochrome P450 (CYP) 3A4. TMR does not inhibit/induce major CYP or uridine diphosphate glucuronosyltransferase (UGT) enzymes and is a P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) substrate. TMR and/or its metabolites inhibit BCRP and organic anion transporter protein 1B1/3 (OATP1B1/3). FTR DDI profile informs coadministration with antiretrovirals (ARV) and other therapeutic classes.MethodsDDI data from 13 studies were compiled to inform the impact of 17 drugs or drug combinations on TMR and the impact of TMR on 15 drugs such as ARVs, rifamycins, opioid substitutes, statins, oral contraceptives (OC), and H2-antagonsits.ResultsFTR with CYP3A4, P-gp, and/or BCRP inhibitors increase TMR concentrations; but, do not pose clinical concern at therapeutic dose. TMR may be administered with weak/moderate inducers with or without coadministration of CYP3A4, P-gp, and/or BCRP inhibitors such as RTV or COBI. Coadministration with strong inducers is contraindicated. FTR may be coadministered with RBT with or without a PK enhancer. However, co-administration of FTR with RIF is contraindicated. FTR can be given with drugs that increase gastric pH; famotidine did not impact TMR PK. TMR may increase concentrations of drugs that are substrates of OATP1B1/3 and BCRP; therefore, most statins require dose reduction (e.g., rosuvastatin dose is limited to ≤10 mg QD). TMR increased EE exposure 40% with no impact on NE; therefore, FTR may be coadministered with OCs containing ≤30 µg EE. TMR had no clinically meaningful impact on TDF, DRV/RTV, ATV/RTV, ATV, RTV, ETR, MET, or BUP/norBUP PK (Table 1).ConclusionFTR can be coadministered with ARVs and most common treatments used to manage HIV co-infections or comorbidities without dose adjustment of either drug except for select HMG-CoA reductase inhibitors and EE-containing OCs. Strong CYP3A inducers are contraindicated. Disclosures All authors: No reported disclosures.